Probing functional HIV-1 envelope glycoprotein conformations with novel potent CD4-mimetic compounds

项目摘要/摘要 人类免疫缺陷病毒(HIV-1)包膜糖蛋白三聚体(gp120-gp41)3与宿主结合 受体，CD4和CCR5/CXCR4，并介导病毒进入靶细胞。CD_4结合诱导 亚稳环境三聚体中的大规模构象变化，导致从预触发的， “封闭”构象向更“开放”构象转变。CD4诱导的Env三聚体的打开允许 Gp120亚基结合CCR5/CXCR4共受体，使病毒和靶细胞膜融合 通过gp41跨膜亚单位。 CD4模拟化合物(CD4mcs)是一种小的有机分子，可以结合到高度保守的口袋( Phe-43空洞)，位于gp120上，靠近CD4结合部位。CD4mcs竞争性地阻断CD4结合和 过早地触发Env的构象变化，类似于由CD4诱导的构象变化。在缺少 共同受体表达的靶细胞，这些过早激活的env迅速和不可逆转地成为非 功能齐全。在亚抑制浓度下，CD4mcs开放Env与抗体的结合，从而 获得中和或细胞毒性潜能。CD4mcs可协同抗体降低HIV-1感染 人源化小鼠的细胞储存库，并保护猴子免受异种SIV粘膜的挑战。 CD4mc支架占据着通往Phe-43腔的gp120“前厅”，是一个关键的决定因素。 这些化合物的抗病毒潜力。更换原型的四甲基哌啶脚手架 带有吲哚支架的CD4mcs打开了合理设计的抗病毒效力改进和 广度。尽管如此，一些具有明显Phe-43空洞的主要HIV-1毒株仍然相对 耐受当前的铅铟CD4mcs。我们最近发现了新型的含有吲哚的cd4mc类似物。 在抗病毒效力和广度方面表现出令人印象深刻的增长的支架。在这项提议中，我们利用 在这一发现上设计了进一步改进的吲哚CD4mcs，以探讨其作用机制 潜在的抗病毒效力，并确定自然的HIV-1环境变异如何影响病毒抵抗力 CD4mcs和病毒复制健康。在这些研究过程中，我们将检验以下假设： 假设1：前庭中吲哚类CD4mcs与保守的gp120元件的接触增加 将进一步增强其抗病毒的效力和广度；假设2：虽然CD4mc耐药性较小 HIV-1毒株的亚群是由Phe-43空洞内的变异引起的，大多数原发HIV-1毒株的敏感性 对CD4mcs的毒株受环境触发能力(环境病毒经历的倾向)的差异控制 构象变化)；和假设3：CD4mcs通过增强与环境三聚体的结合而获得效力， 导致顺序激活和失活，最终导致gp120脱落。 通过检验这些假说所产生的知识将有助于提高抗病毒效果的努力 CD4mcs，并将增强其作为HIV-1包膜构象小分子探针的用途。

PROJECT SUMMARY/ABSTRACT The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer ((gp120-gp41)3) binds to host receptors, CD4 and CCR5/CXCR4, and mediates the entry of the virus into the target cell. CD4 binding induces large-scale conformational changes in the metastable Env trimer, resulting in transitions from a pretriggered, “closed” conformation to more “open” conformations. The CD4-induced opening of the Env trimer allows the gp120 subunit to bind the CCR5/CXCR4 coreceptor, which enables the fusion of viral and target cell membranes by the gp41 transmembrane subunit. CD4-mimetic compounds (CD4mcs) are small organic molecules that bind to a highly conserved pocket (the Phe-43 cavity) on gp120, near the binding site for CD4. CD4mcs competitively block CD4 binding and prematurely trigger conformational changes in Env similar to those induced by CD4. In the absence of a coreceptor-expressing target cell, these prematurely activated Envs rapidly and irreversibly become non- functional. At sub-inhibitory concentrations, CD4mcs open Env to the binding of antibodies that consequently acquire neutralizing or cytotoxic potential. CD4mcs can synergize with antibodies to decrease the HIV-1-infected cell reservoir in humanized mice and to protect monkeys from a heterologous SHIV mucosal challenge. The CD4mc scaffold, which occupies the gp120 “vestibule” leading into the Phe-43 cavity, is a critical determinant of the antiviral potential of these compounds. Replacing the tetramethyl-piperidine scaffold of the prototypic CD4mcs with an indane scaffold opened the door to rationally designed improvements in antiviral potency and breadth. Nonetheless, some primary HIV-1 strains with apparently accessible Phe-43 cavities remain relatively resistant to current lead indane CD4mcs. We have recently identified novel CD4mc analogues with indoline scaffolds that demonstrate impressive increases in antiviral potency and breadth. In this proposal, we capitalize on this discovery to design further improvements in the indoline CD4mcs, to investigate the mechanisms underlying their antiviral potency, and to determine how natural HIV-1 Env variation influences virus resistance to CD4mcs and viral replication fitness. In the course of these studies, we will test the following hypotheses: Hypothesis 1: Increasing the contacts of the indoline CD4mcs with conserved gp120 elements in the vestibule will further enhance their antiviral potency and breadth; Hypothesis 2: Although CD4mc resistance in a small subset of HIV-1 strains results from variation within the Phe-43 cavity, the sensitivity of most primary HIV-1 strains to CD4mcs is governed by differences in Env triggerability (the propensity of the Env to undergo conformational change); and Hypothesis 3: CD4mcs gain potency by enhanced binding to the Env trimer, leading to sequential activation and inactivation that ultimately result in gp120 shedding. The knowledge generated by testing these hypotheses will assist efforts to improve the antiviral efficacy of CD4mcs and will enhance their utility as small-molecule probes of HIV-1 Env conformation.